Vorremmo ringraziare M. Höxter per l&#39;assistenza tecnica di ordinamento primario AECII murino dal livello di biosicurezza 2 campioni. Questo lavoro è stato sostenuto da finanziamenti della Fondazione per la ricerca tedesca (DFG) a DB (SFB587, TP B12 e BR2221/1-1) e una borsa di studio dalla Scuola di Ricerca Biomedica di Hannover (DFG GSC 108) a AA. DB è supportato da Initiative del Presidente e del Fondo di rete dell&#39;Associazione Helmholtz del tedesco Centri di Ricerca (HGF) con W2/W3-029 numero del contratto.

Dettagli relativi reagenti e materiali necessari sono elencati nella tabella alla fine del protocollo di seguito. Prima di iniziare il lavoro, preparare 15 ml provette (uno per topo) contenente 4 ml di dispasi e pre-riscaldare a 37 ° C in un bagno d&#39;acqua. In un blocco riscaldante, poco riscaldare piccole aliquote di 1% agarosio a bassa fusione (in acqua) a 95 ° C fino liquefatto e poi raffreddare a 45 ° C fino all&#39;utilizzo. 1. Preparazione del polmone mouse <ol><li> Sacrifica il mouse CO 2 asfissia. Nota: Non eseguire dislocazione cervicale come questo danneggerà la trachea affinché seguendo fasi del protocollo, come l&#39;installazione del polmone di liquido, non può essere eseguita con successo. Garantire perdita dei riflessi nocicettivi. </li><li> Spruzzare il mouse con etanolo e fare un taglio lungo la linea mediana ventrale del corpo. Tirare il pelo e la pelle ventrale e, successivamente, anche con attenzione tagliare e rimuovere il peritoneo. </li><li> Exsanguinate l&#39;topo tagliando la vena giugulare sinistra e destra (Vena jugularis) e l&#39;arteria renale (renalis Arteria). Rimuovere il sangue che scorre con il tessuto. </li><li> Attenzione puntura e rimuovere il diaframma per esporre il cuore e polmone tagliando via le costole. Fare attenzione a non danneggiare il polmone. </li><li> Con una cannula 26G e una siringa da 10 ml riempita con fosfato fredda tamponata (PBS), forare il ventricolo destro del cuore e la perfusione polmonare con PBS fino libera di sangue. </li><li> Tagliare e rimuovere le ghiandole salivari per esporre la trachea. Anche tagliare con cautela il muscolo che circonda la trachea. </li><li> Inserire una cannula 22G inabitazione nella trachea, rimuovere l&#39;ago e spingere il catetere di plastica verso il polmone. Fissare il catetere legando un piccolo pezzo di filo intorno trachea e il catetere. </li></ol> 2. La digestione enzimatica del tessuto polmonare Se desiderato, un campione di liquido di lavaggio broncoalveolare può esserepreparato dal lavaggio dei polmoni attraverso il catetere inserito nella trachea con PBS o mezzo prima del passaggio successivo. <ol><li> Utilizzando una siringa da 2 ml, accuratamente infondere un volume massimo di 2 ml di dispasi (dal aliquota 4 ml) nel polmone attraverso il catetere in modo che tutti i lobi sono completamente espanso. Scambiare la siringa con una siringa da 1 ml contenente 0,5 ml di agarosio liquefatto e anche instillare nel polmone. </li><li> Lasciare la siringa sul catetere per evitare il riflusso del agarosio e coprire immediatamente il polmone con carta velina laboratorio e ghiaccio. Lascia il gel di agarosio per un paio di minuti. </li><li> Rimuovere la carta velina, ghiaccio, siringa e catetere, tagliare la trachea e le accise il cuore, polmone e del timo dal petto. Risciacquare il polmone in un piatto con PBS e rimuovere il, timo cuore e trachea rimanente. </li><li> Mettere il polmone nelle restanti 2 ml di dispasi e incubare a temperatura ambiente per 45 min. </li></ol> 3. Preparazione del polmoneSospensione cellulare <ol><li> Rimuovere il polmone dal dispasi e trasferirlo in un piatto contenente 7 ml di anticorpo anti-CD16/32 (1 ug / ml concentrazione finale) in DMEM e 100 microlitri DNasi. </li><li> Utilizzando pinze, completamente disintegrare il tessuto polmonare tirandolo a parte e incubare per 10 min a temperatura ambiente mentre agitando lentamente su un agitatore impostato a 200 rpm. Se desiderato, la sospensione cellulare può essere conservato a 4 ° C fino alla fase successiva. </li><li> Filtrare la sospensione cellulare attraverso maglie nylon prima con 100 micron e successivamente con 70 pm, 48 pm e 30 micron pori. Prendono cura di risciacquare ogni maglia nonché i tubi e piatti accuratamente con DMEM per minimizzare la perdita di cellule e massimizzare la resa. Se in comune tutti i campioni, questo può essere realizzato da qui passando successivamente sospensioni cellule da topi di un gruppo attraverso lo stesso filtro. Sostituire il filtro in caso di intasamento. </li><li> A seconda del volume, trasferire il filtrato a uno o più provette da 50 ml ecentrifugare per 15 minuti a 160 xg e 4 ° C. Rimuovere il surnatante. </li><li> Risospendere il pellet di cellule in 2 ml di tampone di lisi degli eritrociti (0,15 M NH 4 Cl, 0,01 M KHCO 3, 0,1 mM EDTA, pH 7,2) e rapidamente terminare lisi mediante aggiunta di 13 ml di terreno DMEM. Trasferire la soluzione in un tubo da 15 ml e centrifugare per 12 min a 160 xg e 4 ° C. </li></ol> 4. Colorazione anticorpi per citometria a flusso Cell-ordinamento <ol><li> Risospendere le cellule in 3 ml di cocktail di anticorpi primari, preparati in DMEM a diluizioni adatte per citometria a flusso. (Anticorpi precedentemente titolata per diluizioni ottimali di lavoro colorando 1 x 10 6 splenociti in un volume di 100 pl. Uso 3 ml di cocktail anticorpo primario contenente le concentrazioni ottimali per ciascuno degli anticorpi utilizzati per le celle pool da cinque topi. Se più topi sono raggruppati per un campione, regolare il volume). </li><li> Per la colorazione, incubare le cellule per 10 minuti al buioa 4 ° C. </li><li> Lavare le cellule riempiendo il tubo 15 ml di DMEM e centrifugazione per 12 min a 160 xg e 4 ° C. </li><li> Nel caso in cui gli anticorpi disaccoppiati o biotinilato sono stati utilizzati in 4.1: Rimuovere il surnatante e risospendere le cellule in 2-3 ml cocktail di anticorpi secondari. Colorazione per 10 min a 4 ° C al buio. </li><li> Lavare le cellule attraverso il tubo di riempimento con DMEM e centrifugazione per 12 min a 160 xg e 4 ° C. </li><li> Risospendere le cellule in 1 ml di DMEM e pre-filtro attraverso un filtro 50 micron in un tubo per cellula-ordinamento. Opzionale: Contare le cellule per ottenere il numero totale delle cellule in sospensione di cellule del polmone. </li></ol> 5. Cell-ordinamento <ol><li> Mescolare le cellule nel vortex prima cernita. Utilizzare un ugello 100 micron per la cella-cernita dei AECII. Pressione del fluido guaina nonché emissione laser e lunghezze d&#39;onda di rilevamento dipendono strumento utilizzato per citometria a flusso di cellule ordinamento nonché i fluorocromi usati nella antibody colorazione procedura. </li><li> Porta sulle cellule SSC alti che sono negativi per i fluorocromi utilizzati per la colorazione e l&#39;ordinamento in una provetta contenente DMEM. Utilizzare in avanti altezza scatter (FSC-H) vs area (FSC-A) e l&#39;altezza dispersione laterale (SSC-H) vs area (SSC-A) di Windows per escludere eventuali doppiette o aggregati cellulari (vedi dettagli della strategia di gating in Figura 1 a). </li><li> Per raccogliere la centrifuga ordinati cellule per 20 minuti a 280 xg e 4 ° C. </li><li> Risospendere le cellule in coltura-medio o tampone come richiesto per la successiva analisi. </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+epithelium:+structural+and+functional+properties+in+health+and+disease.">The airway epithelium: structural and functional properties in health and disease.</a> Respirology. 8, 432-446 (2003).</li><li>Schmiedl, A., Kerber-Momot, T., Munder, A., Pabst, R., Tschernig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+distribution+in+lung+parenchyma+early+after+pulmonary+infection+with+Pseudomonas+aeruginosa.">Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa.</a> Cell Tissue Res. 342, 67-73 (2010).</li><li>Loveday, E. K., Svinti, V., Diederich, S., Pasick, J., Jean, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal-+and+Strain-Specific+Host+MicroRNA+Molecular+Signatures+Associated+with+Swine-Origin+H1N1+and+Avian-Origin+H7N7+Influenza+A+Virus+Infection.">Temporal- and Strain-Specific Host MicroRNA Molecular Signatures Associated with Swine-Origin H1N1 and Avian-Origin H7N7 Influenza A Virus Infection.</a> J. Virol. 86, 6109-6122 (2012).</li><li>Marriott, H. 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Immun. 80, 1140-1149 (2012).</li><li>Mata, M., Morcillo, E., Gimeno, C., Cortijo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetyl-L-cysteine+(NAC)+inhibit+mucin+synthesis+and+pro-inflammatory+mediators+in+alveolar+type+II+epithelial+cells+infected+with+influenza+virus+A+and+B+and+with+respiratory+syncytial+virus+(RSV).">N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV).</a> Biochem. Pharmacol. 82, 548-555 (2011).</li><li>Zarbock, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surfactant+protein+C+mutation+A116D+alters+cellular+processing,+stress+tolerance,+surfactant+lipid+composition,+and+immune+cell+activation.">The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.</a> BMC. Pulm. Med. 12, 15 (2012).</li><li>Taubenberger, J. K., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+influenza+virus+infections.">The pathology of influenza virus infections.</a> Annu. Rev. Pathol. 3, 499-522 (2008).</li><li>Dobbs, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+alveolar+type+II+cells.">Isolation and culture of alveolar type II cells.</a> Am. J. Physiol. 258, L134-L147 (1990).</li><li>Corti, M., Brody, A. R., Harrison, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+murine+alveolar+type+II+cells.">Isolation and primary culture of murine alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 14, 309-315 (1996).</li><li>Beers, M. F., Kim, C. Y., Dodia, C., Fisher, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+synthesis,+and+processing+of+surfactant+protein+SP-C+in+rat+lung+analyzed+by+epitope-specific+antipeptide+antibodies.">Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies.</a> J. Biol. Chem. 269, 20318-20328 (1994).</li><li>Phelps, D. S., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+pulmonary+surfactant+proteins+using+immunohistochemistry+and+tissue+in+situ+hybridization.">Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization.</a> Exp. Lung Res. 17, 985-995 (1991).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+human+alveolar+type+II+cells+secrete+antiviral+IL-29+(IFN-lambda+1)+in+response+to+influenza+A+infection.">Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection.</a> J. Immunol. 182, 1296-1304 (2009).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+response+to+influenza+A+virus+in+differentiated+human+alveolar+type+II+cells.">Innate immune response to influenza A virus in differentiated human alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 45, 582-591 (2011).</li></ol>

Nessun conflitto di interessi dichiarati.

Nostro protocollo per l&#39;isolamento di AECII murino mediante citometria a flusso offre un modo rapido di accedere cellule primarie del polmone mouse per tutta una serie di studi funzionali e molecolari. La procedura descritta produce popolazioni altamente vitali e puro di AECII che sono in numero sufficiente per le successive analisi dirette, come l&#39;RNA isolamento (vedi figura 2b) e studi del trascrittoma. Per applicazioni funzionali, è anche possibile per coltivare le cellule isolate, consenten...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> inabitazione cannula Introcan 22G </td><td> Braun </td><td> REF 4252098B </td><td></td></tr><tr><td> Dispasi, 100 ml (5000 unità caseinolytic) </td><td> BD Biosciences </td><td> 354235 </td><td> aliquota da 4 ml in provette da 15 ml, conservare a -20 ° C </td></tr><tr><td> Biozym placca agarosio </td><td> Biozym </td><td> 840101 </td><td> 1% w / v in H 2 O </td></tr><tr><td> Deoxyribonuclease I da pancreas bovino, 2000 unità Kunitz / flaconcino </td><td> Sigma-Aldrich </td><td> D4263 </td><td> fresco Sciogliere il contenuto di 1 fiala in 300 ml DMEM </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 22320-022 </td><td> utilizzato secondo quanto previsto dal costruttore (Low Glucose, piruvato, HEPES) </td></tr><tr><td> filtri cellulari (100 micron, 75 micron) </td><td> BD Falcon </td><td> 352360, 352350 </td><td></td></tr><tr><td> maglia di nylon (48 micron, 30 micron) </td><td> Buckmann GmbH </td><td> 03-48/26-1020, 03-30/18-108 </td><td></td></tr><tr><td> CellTrics 50 um filtro </td><td> PARTEC </td><td> 04-0042-2317 </td><td></td></tr><tr><td> anti-topo CD16/CD32 </td><td> BioLegend </td><td> 101302 </td><td> clone 93; purificato </td></tr><tr><td> anti-topo F4/80 </td><td> BioLegend </td><td> 123116 </td><td> clone BM8; APC accoppiato </td></tr><tr><td> anti-topo CD11b </td><td> BioLegend </td><td> 101208 </td><td> clone M1/70; PE accoppiato </td></tr><tr><td> anti-topo CD11c </td><td> BioLegend </td><td> 117310 </td><td> clone N418; APC accoppiato </td></tr><tr><td> anti-topoCD45 </td><td> BioLegend </td><td> 103102 </td><td> clone 30-F11; purificato </td></tr><tr><td> anti-topo CD19 </td><td> eBioscience </td><td> 12-0193-83 </td><td> EBIO 1D3; PE accoppiato </td></tr><tr><td> policlonale di capra anti-topo IgG </td><td> BD Pharmingen </td><td> 550767 </td><td> policlonale, PE accoppiato </td></tr><tr><td></td><td></td><td></td><td> Anticorpi accoppiati a fluorocromi alternativi possono essere utilizzati, a seconda del citofluorimetro e laser disponibili. </td></tr></tbody></table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

Durante gli ultimi anni, il contributo delle cellule alveolari di tipo II epiteliali (AECII) a vari aspetti della regolazione immunitaria nel polmone è stato sempre più riconosciuto. AECII hanno dimostrato di partecipare alla produzione di citochine in vie aeree infiammate ed agire anche come cellule presentanti l&#39;antigene sia infezione e cellule T mediata autoimmunità 1-8. Pertanto, essi sono particolarmente interessanti anche in contesti clinici, quali vie aeree iper-reattività estera e di auto-antigeni così come le infezioni che direttamente o indirettamente si rivolgono AECII. Tuttavia, la nostra comprensione delle funzioni immunologiche dettagliate serviti da cellule alveolari di tipo II epiteliali nel polmone sano così come in infiammazione rimane frammentario. Molti studi in materia di funzione AECII vengono eseguite utilizzando il mouse o umani linee cellulari epiteliali alveolari 9-12. Lavorando con linee cellulari offre sicuramente una serie di vantaggi, come la disponibilità di grandi numeri of cellule per analisi approfondite. Tuttavia, riteniamo che l&#39;uso di primaria AECII murino permette una migliore comprensione del ruolo di questo tipo di cellule in processi complessi come infezione o infiammazione autoimmune. Primaria AECII murino possono essere isolati direttamente da animali affetti da tali disturbi respiratori, nel senso che sono stati sottoposti a tutti gli altri fattori estrinseci che giocano un ruolo nel contesto analizzato. Come esempio, AECII vitali possono essere isolate da topi infettati intranasale con virus influenzale A, che colpisce principalmente queste cellule per la replicazione 13. È importante sottolineare che, attraverso l&#39;infezione ex vivo di AECII isolati da topi sani, gli studi sulle risposte cellulari montati su di infezione può essere ulteriormente estesa. Nostro protocollo per l&#39;isolamento di primaria AECII murino si basa sulla digestione enzimatica del polmone topo seguita da etichettare la sospensione cellulare risultante con anticorpi specifici per CD11c, CD11b, F4/80,CD19, CD45 e CD16/CD32. AECII granulari vengono identificate come la dispersione non marcato e lateralmente alta (SSC alta) popolazione cellulare e sono separati da fluorescenza activated cell smistamento 3. In confronto ai metodi alternativi di isolare cellule epiteliali primarie da polmoni di topo, il nostro protocollo per l&#39;isolamento di citometria a flusso AECII per selezione negativa produce intatta, altamente puro AECII vitale e in tempi relativamente brevi. Inoltre, e in contrasto con i metodi convenzionali di isolamento di panning e deplezione dei linfociti tramite legame di anticorpo-accoppiati biglie magnetiche 14, 15, citometria a flusso di cellule ordinamento consente discriminazione attraverso dimensione cellulare e granularità. Dato che la strumentazione per selezione cellulare citofluorimetrica è disponibile, la procedura descritta può essere applicata a costi relativamente bassi. Accanto a anticorpi standard e enzimi per la disintegrazione del polmone, senza reagenti aggiuntivi, come magneticoperline sono richiesti. Le cellule isolate sono adatti per un&#39;ampia gamma di studi funzionali e molecolari, che comprendono la coltura in vitro di cellule T e saggi di stimolazione così come trascrittoma, proteoma o analisi secretoma 3, 4.

Quando si ordina sospensioni di cellule polmonari isolati da topi sani, il cancello AECII in genere rappresentano circa il 42 ± 10% di tutti gli eventi. Tale percentuale può essere notevolmente inferiore quando topi con patologie respiratorie come una infezione virale vengono utilizzati, come la sospensione cellulare iniziale conterrà una percentuale notevolmente più elevata di linfociti e di altre cellule immunitarie reclutati per le vie respiratorie. Per AECII isolata da polmoni IAV infetti al giorno 3 dopo l&#39;...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Descriviamo l&#39;isolamento rapido di primarie murine di tipo II le cellule epiteliali alveolari (AECII) per selezione di citometria di flusso negativo. Questi AECII mostrano alta vitalità e purezza e sono adatte per una vasta gamma di studi funzionali e molecolari riguardanti il ​​loro ruolo in condizioni respiratorie come le malattie autoimmuni o infettive.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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Immunology and Infection

26.1K Views.  Helmholtz Centre for Infection Research. Descriviamo l'isolamento rapido di primarie murine di tipo II le cellule epiteliali alveolari (AECII) per selezione di citometria di flusso negativo. Questi AECII mostrano alta vitalità e purezza e sono adatte per una vasta gamma di studi funzionali e molecolari riguardanti il ​​loro ruolo in condizioni respiratorie come le malattie autoimmuni o infettive.

Video: Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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Immunologia e infezioni

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Analisi del danno epiteliale Prodotto da<em&gt; Entamoeba histolytica</em&gt; Infezione

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolamento e analisi citofluorimetrica di-Glioma infiltrazione sangue periferico mononucleari Cellule

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolamento e caratterizzazione citometria a flusso di Murine Piccolo intestinale Linfociti

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

L&#39;isolamento e la citometria a flusso Analisi di Human endocervicale Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Digestione del fegato murino per un'analisi citofluorimetrica delle cellule endoteliali linfatiche

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...